1. Field of the Invention
The invention relates to methods and reagents for preparing multifunctional probes, and more particularly to multifunctional probes that are synthesized in a single step using peptide scaffold-based multifunctional single-attachment-point reagents.
2. Description of the Related Art
The design of new nanomaterials with ever higher levels of multifunctional capability, and consequent chemical complexity, is a common challenge to the fields of drug delivery, in vivo molecular imaging, and sensor development (see, e.g., Ferrari, Nat Rev Cancer (2005) 5:161-171; Rhyner et al., 110 Nanomed (2006) 1: 209-217; and Cai et al., Small (2007) 3:1840-1854). Multifunctionality is relatively easy to achieve with nanoparticles or polymers that afford a large number of similar reactive sites. Multifunctional probes can then be obtained by reacting a substrate with two or more monofunctional reagents each with a reactive group sequentially, see FIG. 1a. Examples of sequential modification strategies abound with quantum dots (see, Gao et al., Nat Biotechnol (2004) 22:969-976), gold and magnetic iron oxide nanoparticles (see, Weissleder et al., 115 Nat Biotechnol (2005) 23:1418-1423; and Kumar et al., Nat Protoc (2008) 3:314-320); and polymers like polylysines (see Weissleder et al., Nat Biotechnol (1999) 17:375-378).
However, sequential syntheses of multifunctional probes allow the stoichiometric ratios of functional groups to vary since each group is attached independently. As the number of functional groups increases from two to three or more, the problem of non-stoichiometric functional group attachment escalates. When multifunctional probes are considered for clinical use, a frequent rationale for animal experiments, fixed functional group ratios in each preparation becomes a necessity. In addition, the extent of substrate modification with non-light absorbing functional groups (e.g. chelate, biotin, polymer) sometimes cannot be determined by simple analytical procedures. Yet another limitation of multifunctional probe syntheses using multiple reagents occurs when they are considered for substrates possessing a single reactive center, a situation more frequently encountered as substrate size decreases from nanoparticles (>500 kDa) to small macromolecules (5-50 kDa, e.g. small proteins) or low molecular weight molecules (<5 kDa, e.g. drugs, peptides, hormones). Addition of multiple functional groups is also impossible with substrates like annexin V (32 kDa) which, though affording multiple reactive amines, loses activity after modification of a single amine (see, Schellenberger et al., 120 Chembiochem (2004) 5:275-279).
Therefore, there is need for methods and reagents for preparing multifunctional probes such that the multifunctional probes can have controlled stoichiometric ratios of functional groups on the probe.